Mitochondria, cellular organelles present in all known eukaryotic cells, contain a semi-autonomous genetic system -- a unique quality among metazoans. In mammalian cells, there is a marked conservation of genetic function and arrangement within the mitochrondrial DNA (mtDNA) molecule, which is completely and symmetrically transcribed, and encodes either proteins or the protein-synthesizing apparatus of the mitochrondria. The remaining portion of the mtDNA contains the origin of DNA replication and the only two known origins of transcription. Moreover, there is economy of nucleotide base sequence within a species, although intraspecific variability does exist as seen by detailed restriction endonuclease analyses. Other investigators have located the greatest degree of polymorphism to the region which contains the replicational and transcriptional control sequences. It has been our goal to characterize this variability at the level of nucleotide sequence. We have cloned this polymorphic region of human mtDNA isolated from several different individual placentas, performed restriction mapping and hybridization analyses to define the precise origin of replication within the cloned segments, and the variable regions were conformed by determination of the DNA sequence.